Up-conversion fluorescence sensor, construction method thereof and application of up-conversion fluorescence sensor in detection of parathion-methyl in rhizoma polygonati

Abstract

The invention discloses an up-conversion fluorescence sensor, a construction method thereof and application of the up-conversion fluorescence sensor in detection of parathion-methyl in rhizoma polygonati, and belongs to the field of pesticide detection. The upconversion fluorescence sensor comprises an upconversion nanometer material modified by a parathion-methyl aptamer and a Fe3O4 (at) MoS2 nanometer composite material modified by cDNA (complementary deoxyribonucleic acid), and nucleotide sequences of the parathion-methyl aptamer and the cDNA are complementarily paired. When parathion-methyl in rhizoma polygonati is detected by using the fluorescence sensor, when parathion-methyl exists in a system, the aptamer on the surface of the up-conversion nano material has a specific recognition effect on parathion-methyl, and parathion-methyl can be preferentially captured through the change of spatial conformation; and the content of parathion-methyl is detected by virtue of the change of fluorescence intensity. The method for detecting parathion-methyl in polygonatum kingianum by using the up-conversion fluorescence sensor is high in sensitivity, good in specificity and high in accuracy.

Description

Technical Field

[0001] The present invention relates to the field of pesticide detection, and particularly to an upconversion fluorescence sensor, a construction method thereof, and an application thereof in detecting methyl parathion in Polygonatum sibiricum. Background Art

[0002] Polygonatum sibiricum (Ps) is a plant of the genus Polygonatum in the family Liliaceae. Its rhizome is a traditional Chinese medicine material with the effects of invigorating the spleen, moistening the lungs and promoting fluid production. At present, Polygonatum sibiricum is mainly interplanted under forests. However, with the increasing market demand year by year, its cultivation methods are gradually diversified. However, during the cultivation process, the use of pesticides to prevent and control pests and diseases inevitably leads to the problem of pesticide residues, which not only endangers the health of consumers, but also restricts the sustainable development of the Polygonatum sibiricum industry. Therefore, it is of great significance to establish a rapid, simple and efficient pesticide residue detection technology to effectively monitor the pesticide residues in the production, processing, storage and transportation links of Polygonatum sibiricum.

[0003] Methyl parathion (MP) is a broad-spectrum organophosphorus pesticide, which is used in the production of Polygonatum sibiricum to control pests and increase the yield of Polygonatum sibiricum. However, its irregular use often results in its residue in Polygonatum sibiricum, and its residue amount may exceed the maximum residue limit stipulated by the state. The residual methyl parathion may ultimately enter the human body through the food chain, thereby causing damage to the human nervous system and producing poisoning reactions, etc. At present, the methods for detecting methyl parathion include gas chromatography, enzyme-linked immunosorbent assay, etc. Although these methods have their own advantages, they also have disadvantages such as cumbersome detection processes, long detection times, high analysis costs, and the need for professional personnel to operate to complete the detection. Therefore, there is an urgent need for a simple, accurate, sensitive and rapid analysis method for detecting methyl parathion in Polygonatum sibiricum to meet the market demand. Summary of the Invention

[0004] The object of the present invention is to provide an upconversion fluorescence sensor, a construction method thereof, and an application thereof in detecting methyl parathion in Polygonatum sibiricum, so as to solve the problems existing in the above-mentioned prior art. An upconversion fluorescence sensor constructed by combining an upconversion nanomaterial modified with a methyl parathion aptamer (UCNPs-Apt) and an Fe3O4@MoS2 nanocomposite modified with cDNA (Fe3O4@MoS2-cDNA) gathers the strong fluorescence and specific recognition function of UCNPs-Apt, as well as the excellent magnetism, large specific surface area of Fe3O4@MoS2-cDNA, and the complementary pairing of cDNA and aptamer. It improves the response speed, specificity and sensitivity of methyl parathion detection, simplifies the detection procedure, saves the detection cost, and realizes the accurate detection of methyl parathion in Polygonatum sibiricum.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an upconversion fluorescence sensor for detecting methyl parathion in polygonatum sibiricum. The upconversion fluorescence sensor includes an upconversion nanomaterial modified with a methyl parathion aptamer and an Fe3O4@MoS2 nanocomposite modified with cDNA. Among them, the nucleotide sequences of the methyl parathion aptamer and the cDNA are complementary and paired, and the upconversion nanomaterial is a rare earth upconversion nanomaterial doped with gadolinium, ytterbium and / or erbium.

[0007] Preferably, the nucleotide sequence of the methyl parathion aptamer is as shown in SEQ ID NO: 1, and the nucleotide sequence of the cDNA is as shown in SEQ ID NO: 2.

[0008] The present invention also provides a preparation method of an upconversion fluorescence sensor for detecting methyl parathion in polygonatum sibiricum, including the following steps:

[0009] (1) Dissolve polyethyleneimine in ethylene glycol, then add gadolinium chloride hexahydrate, sodium chloride, erbium chloride anhydrous and ytterbium chloride hexahydrate to react to obtain a mixed solution. Dissolve ammonium chloride in ethylene glycol and then drop it into the mixed solution to continue the reaction, and then transfer it to a reaction kettle for high-temperature reaction to obtain an amino-modified upconversion nanomaterial;

[0010] (2) Mix ferric chloride, anhydrous sodium acetate and ethylene glycol to react to obtain Fe3O4 nanoparticles. Then ultrasonically disperse the Fe3O4 nanoparticles in distilled water, add ammonium molybdate tetrahydrate and thiourea for high-temperature reaction to obtain an Fe3O4@MoS2 nanocomposite; Mix the Fe3O4@MoS2 nanocomposite with 3-aminopropyltriethoxysilane in an ethanol solution and oscillate to react to obtain an amino-modified Fe3O4@MoS2;

[0011] (3) Mix the amino-modified upconversion nanomaterial with avidin and incubate with oscillation to obtain an avidin-modified upconversion nanomaterial; Then incubate the avidin-modified upconversion nanomaterial with a biotin-modified methyl parathion aptamer with oscillation to obtain a methyl parathion aptamer-modified upconversion nanomaterial;

[0012] (4) Mix the amino-modified Fe3O4@MoS2 with avidin and incubate with oscillation to obtain an avidin-modified Fe3O4@MoS2; Then incubate the avidin-modified Fe3O4@MoS2 with a biotin-modified cDNA with oscillation to obtain a cDNA-modified Fe3O4@MoS2 nanocomposite;

[0013] (5) Mix and incubate the upconversion nanomaterials modified with methyl parathion aptamer and the cDNA-modified Fe3O4@MoS2 nanocomposites to obtain an upconversion fluorescence sensor for detecting methyl parathion in polygonatum sibiricum.

[0014] Preferably, in step (1), the dosage ratio of the polyethyleneimine, ethylene glycol, gadolinium chloride hexahydrate, sodium chloride, erbium chloride anhydrous, and ytterbium chloride hexahydrate is 500 mg: 35 mL: 361.1 mg: 58.5 mg: 13.3 mg: 76.4 mg, and the reaction conditions are: stirring for 20 - 30 min;

[0015] The dosage ratio of the ammonium fluoride and ethylene glycol is 296.3 mg: 15 mL, and the reaction conditions are: stirring for 8 - 12 min;

[0016] The conditions for the high-temperature reaction in the reaction kettle are: reacting at 180 - 200 °C for 10 - 12 h.

[0017] Preferably, in step (2), the dosage ratio of the ferric chloride, sodium acetate anhydrous, and ethylene glycol is 0.5 g: 3.0 g: 30 mL, and the reaction conditions are: reacting at 180 - 190 °C for 4 h;

[0018] The dosage ratio of the Fe3O4 nanoparticles, ammonium molybdate tetrahydrate, and thiourea is 30 mg: 0.8 g: 2.0 g, and the high-temperature reaction conditions are: reacting at 180 - 190 °C for 12 h;

[0019] The dosage ratio of the Fe3O4@MoS2 nanocomposites and 3-aminopropyltriethoxysilane is 40 mg: 500 μL, and the conditions for the oscillation reaction are: oscillating and reacting at 60 °C for 12 h.

[0020] Preferably, in step (3), the temperature for mixing and incubating the amino-modified upconversion nanomaterials and avidin is 37 °C, and the time is 12 h;

[0021] The temperature for mixing and incubating the avidin-modified upconversion nanomaterials and biotin-modified methyl parathion aptamer is 37 °C, and the time is 6 h;

[0022] The nucleotide sequence of the biotin-modified methyl parathion aptamer is as shown in SEQ ID NO: 1.

[0023] Preferably, in step (4), the temperature for mixing and incubating the amino-modified Fe3O4@MoS2 and avidin is 37 °C, and the time is 12 h;

[0024] The temperature for mixing and incubating the avidin-modified Fe3O4@MoS2 and biotin-modified cDNA is 37 °C, and the time is 6 h;

[0025] The nucleotide sequence of the biotin-modified cDNA is shown in SEQ ID NO: 2.

[0026] Preferably, in step (5), the volume ratio of the methyl parathion aptamer-modified upconversion nanomaterial to the cDNA-modified Fe3O4@MoS2 nanocomposite is 5:3, the concentration of the methyl parathion aptamer-modified upconversion nanomaterial is 1 mg / mL, the concentration of the cDNA-modified Fe3O4@MoS2 nanocomposite is 0.8 mg / mL, and the conditions for the mixed incubation are: reacting at 37 °C for 30 min.

[0027] The present invention also provides the application of the above upconversion fluorescence sensor in detecting methyl parathion in polygonatum sibiricum.

[0028] The present invention also provides a method for detecting methyl parathion in polygonatum sibiricum, which includes the steps of mixing the above upconversion fluorescence sensor with a polygonatum sibiricum sample to be detected for reaction, and obtaining the content of methyl parathion in polygonatum sibiricum by detecting the upconversion fluorescence intensity value at 540 nm.

[0029] In the embodiment of the present invention, the following method is specifically adopted to detect methyl parathion in polygonatum sibiricum:

[0030] (1) Establishment of the methyl parathion content standard curve:

[0031] Mix the methyl parathion aptamer-modified upconversion nanomaterial (UCNPs-Apt) and the cDNA-modified Fe3O4@MoS2 nanocomposite (Fe3O4@MoS2-cDNA) for reaction to obtain an upconversion fluorescence sensing detection system, collect the fluorescence spectrum. After continuously reacting by adding methyl parathion standard solutions with different concentrations to the detection system, separate and wash the mixture with a magnet and resuspend it in a buffer solution, and collect the fluorescence spectrum under the excitation of a 980 nm exciter. Construct a standard curve with the logarithm of the methyl parathion concentration as the abscissa and the relative upconversion fluorescence intensity value at 540 nm of the fluorescence spectrum characteristic peak as the ordinate;

[0032] (2) Detection of the methyl parathion content in polygonatum sibiricum:

[0033] Perform pretreatment on the polygonatum sibiricum sample, extract methyl parathion to obtain a methyl parathion solution to be detected; add the methyl parathion solution to be detected to the upconversion fluorescence sensing detection system obtained in step (1), and after reaction, collect the fluorescence spectrum to obtain the upconversion fluorescence intensity value at 540 nm, and substitute it into the standard curve obtained in step (1) to calculate the content of methyl parathion in polygonatum sibiricum.

[0034] Preferably, in step (1), the concentration of UCNPs-Apt is 1 mg / mL; the concentration of Fe3O4@MoS2-cDNA is 0.8 mg / mL; the volume ratio of UCNPs-Apt, Fe3O4@MoS2-cDNA and the methyl parathion solution to be detected is 5:3:2.

[0035] Preferably, in step (1), the mixing reaction temperature of UCNPs-Apt and Fe3O4@MoS2-cDNA is 37 °C and the time is 30 min; the reaction temperature after adding methyl parathion standard solutions with different concentrations to the detection system is 37 °C and the time is 30 min.

[0036] Preferably, in step (2), the extraction method of the methyl parathion solution to be detected is as follows: adding a methyl parathion standard product with a known concentration to polygonatum sibiricum powder and mixing. Mixing the above mixed sample with acetonitrile, vortex extracting, centrifuging and removing solids, repeating the above steps, and combining the supernatant. Rotating and evaporating the supernatant at 80 °C to evaporate the acetonitrile solvent until almost dry. Diluting the dried residue to a constant volume and vortex mixing.

[0037] Preferably, in step (2), the volume ratio of the upconversion fluorescence sensing detection system to the methyl parathion solution to be detected and the reaction conditions are the same as those in step (1).

[0038] The present invention discloses the following technical effects:

[0039] (1) The present invention discloses an upconversion fluorescence sensor, a construction method thereof and an application in detecting methyl parathion in polygonatum sibiricum, explores a new fluorescence sensing method for detecting methyl parathion in polygonatum sibiricum, focuses on breaking through the problem of controllable synthesis of UCNPs-Apt and Fe3O4@MoS2-cDNA, and constructs an upconversion fluorescence sensor to achieve efficient detection of methyl parathion in the complex matrix of polygonatum sibiricum.

[0040] (2) For the upconversion fluorescence sensor constructed by the present invention, the fluorescence signal material used is UCNPs, and its luminescence process mainly occurs in the matrix material, which is free from the influence of the external environment, has stable photochemical properties. At the same time, UCNPs can effectively avoid the influence of the autofluorescence of complex sample matrices during the detection process, enhancing the detection sensitivity and accuracy; synthesizing UCNPs modified with methyl parathion aptamer (UCNPs-Apt), which can specifically bind to methyl parathion. When methyl parathion is present, the Apt on the surface of UCNPs has a specific recognition effect on methyl parathion, and will preferentially capture methyl parathion through the change of spatial conformation, and detect the content of methyl parathion by means of the change of fluorescence intensity, improving the detection specificity.

[0041] (3) The upconversion fluorescence sensor constructed in the present invention also uses Fe3O4@MoS2. This material not only has good magnetism and can quickly and effectively achieve the separation purpose without centrifugal filtration, but also has a large specific surface area, which can meet the requirement of modifying a large number of complementary strands cDNA of methyl parathion aptamer on its surface to prepare Fe3O4@MoS2-cDNA. Compared with the traditional Fe3O4-cDNA, Fe3O4@MoS2-cDNA can fully form a UCNPs-Apt-cDNA-Fe3O4@MoS2 hybrid detection system with UCNPs-Apt through the base complementary pairing of Apt-cDNA. Even if a trace amount of methyl parathion extracted from polygonatum sibiricum is added to the system, it can quickly cause a response of the fluorescence signal, so as to realize the rapid detection of trace pollutants in the complex sample of polygonatum sibiricum, shorten the detection time and improve the detection efficiency.

[0042] (4) The new fluorescence sensing method for detecting methyl parathion in polygonatum sibiricum of the present invention has a wide detection range (0.01 - 100 ng / mL), and the detection limit is as low as 0.002 ng / mL. Comparing the constructed new fluorescence sensing method with the traditional ELISA method, there is no significant difference, which can meet the highly sensitive detection of the methyl parathion content in polygonatum sibiricum, and can provide a theoretical basis for the real-time monitoring of methyl parathion in the actual detection process. Description of the Drawings

[0043] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0044] Figure 1 It is the preparation process and detection principle diagram of the upconversion fluorescence sensor of the present invention;

[0045] Figure 2 It is the characterization diagram of the upconversion nanomaterial; where A is the transmission electron microscope image, B is the X-ray diffraction spectrum, C is the Fourier transform infrared spectrum, and D is the fluorescence spectrum;

[0046] Figure 3 It is the characterization diagram of Fe3O4@MoS2; where A is the scanning electron microscope image, B is the X-ray diffraction spectrum, and C is the Fourier transform infrared spectrum;

[0047] Figure 4It is the UV-vis absorption spectrogram; among which, A is the UV-vis absorption spectrogram of UCNPs and UCNPs-Apt, and B is the UV-vis absorption spectrogram of Fe3O4@MoS2 and Fe3O4@MoS2-cDNA;

[0048] Figure 5 It is the optimization of the detection system of the upconversion fluorescence sensor of the present invention; among which, A is the optimization of the concentration of Fe3O4@MoS2-cDNA, B is the optimization of the incubation time of UCNPs-Apt and Fe3O4@MoS2-cDNA, and C is the optimization of the binding time of the aptamer and parathion-methyl;

[0049] Figure 6 It is the analysis diagram of the detection sensitivity of the upconversion fluorescence sensor of the present invention; among which, A is the upconversion fluorescence spectrogram for detecting different concentrations of parathion-methyl, and B is the standard curve diagram established after detecting different concentrations of parathion-methyl;

[0050] Figure 7 It is the analysis diagram of the detection specificity of the upconversion fluorescence sensor of the present invention; among which, A is the fluorescence intensity of the detection system at 540 nm when there is only one pesticide, and B is the fluorescence intensity of the detection system at 540 nm when two pesticides exist simultaneously. Detailed implementation manners

[0051] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be regarded as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0052] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0053] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0054] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are merely exemplary.

[0055] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0056] The schematic diagram of the upconversion fluorescence sensor constructed by the present invention for the detection of parathion-methyl in polygonatum sibiricum is shown in Figure 1 . The specific construction and detection processes are further described in the examples.

[0057] Example 1 Construction method of upconversion fluorescence sensor

[0058] (1) Preparation of amino-modified upconversion nanomaterials

[0059] Dissolve polyethyleneimine (PEI, 0.5 g) in ethylene glycol (EG, 35 mL), then add 361.1 mg GdCl3·6H2O, 58.5 mg NaCl, 13.3 mg ErCl3, and 76.4 mg YbCl3·6H2O, and stir vigorously for 30 min to obtain a mixed solution. Gradually add 15 mL of EG in which 296.3 mg of ammonium fluoride (NH4F) is dissolved dropwise to the above mixed solution, stir for 10 min, transfer to a reaction kettle, and react at 200 °C for 12 h. After the reaction is completed, wait for the reaction kettle to cool, centrifuge to collect the precipitate, wash with 50% ethanol solution, and the product obtained after centrifugal drying is the amino-modified upconversion nanomaterial (UCNPs).

[0060] Observation of the amino-modified upconversion nanomaterial (UCNPs-NH2) by transmission electron microscopy, the results are as shown in Figure 2 A in it. The synthesized material has good dispersibility, the average particle size is about 100 nm, and the appearance is approximately hexagonal.

[0061] The X-ray diffraction spectrum is as shown in Figure 2 B in it. The diffraction peaks of the upconversion nanomaterial coincide with the standard JCPDS card (No. 27-0699) of β-NaGdF4, indicating that the prepared nanomaterial has the structural characteristics of a hexagonal crystal phase.

[0062] The infrared spectrum is as shown in Figure 2 C in it. At 3340 cm -1 , 2930 cm -1 , 2856 cm -1 , 1633 cm -1 and 1399 cm-1 There are five characteristic peaks. Among them, the characteristic peaks at 3340 cm -1 and 1633 cm -1 are attributed to the stretching and bending vibration peaks of N-H; the characteristic peaks at 2930 cm -1 and 2856 cm -1 are the asymmetric and symmetric stretching vibration peaks of -CH2- in the long alkyl chain; in addition, the characteristic peak at 1399 cm -1 is the stretching vibration peak of the C-N bond. The appearance of these characteristic peaks indicates that the amino group has been modified on the surface of UCNPs.

[0063] The fluorescence spectrum is shown in Figure 2 D as shown, and three significant characteristic emission peaks are observed at 520 nm, 540 nm, and 654 nm, corresponding to the Er 3+ energy level transitions 2 H 11 / 2 → 4 I 15 / 2 , 4 S 3 / 2 → 4 I 15 / 2 and 4 F 9 / 2 → 4 I 15 / 2 , which confirms that the prepared upconversion nanomaterials have good fluorescence properties.

[0064] The above characterization results prove that the synthesis of amino-modified upconversion nanomaterials is successful.

[0065] (2) Preparation of Amino-Modified Fe3O4@MoS2

[0066] First, Fe3O4 nanoparticles were synthesized: 0.5 g of ferric chloride (FeCl3), 3.0 g of anhydrous sodium acetate, and 30 mL of ethylene glycol were mixed, and stirred vigorously at 50 °C until the solution became clear. Subsequently, it was transferred to a reaction kettle and reacted at 190 °C for 4 h. After the reaction, the product was collected by magnetic separation, washed 3 times with 50% ethanol solution, and dried at 60 °C to obtain Fe3O4 nanoparticles.

[0067] Then, Fe3O4@MoS2 was prepared: 30 mg of the synthesized Fe3O4 nanoparticles were dispersed in 30 mL of distilled water. After ultrasonic dispersion, 0.8 g of ammonium molybdate tetrahydrate ((NH4)6Mo7O 24 ·4H2O) and 2.0 g of thiourea were added in sequence, and ultrasonic treatment was continued. Subsequently, the mixture was placed in a stainless steel autoclave lined with polytetrafluoroethylene and reacted at 180 °C for 12 hours. After the reaction, the product was also collected by magnetic separation, washed 3 times with 50% ethanol solution, and dried at 60 °C to obtain Fe3O4@MoS2. The scanning electron microscope image is shown inFigure 3 As shown in A, there is an obvious "pompon chrysanthemum" structure on the surface of the Fe3O4@MoS2 composite material, and the average particle size of the material is about 320 nm; the X-ray diffraction spectrogram is as Figure 3 shown in B. The diffraction peaks of Fe3O4 nanoparticles coincide with the PDF standard card of Fe3O4 (PDF#74-0748), indicating that it has a cubic phase structure. The two broad peaks (100) and (110) are consistent with the PDF standard card of 2H-MoS2 (PDF#37-1492). Typical diffraction peaks of Fe3O4 and MoS2 can be observed in the XRD pattern of Fe3O4@MoS2; the above characterization results prove that the Fe3O4@MoS2 material is successfully prepared.

[0068] Finally, amino modification is carried out: 40 mg of Fe3O4@MoS2 is mixed with 500 μL of 3-aminopropyltriethoxysilane (APTES) in 10 mL of ethanol solution, and oscillated at 60 °C for 12 hours. After the reaction, it is collected by magnetic separation. After washing the product with 50% ethanol, it is dried to obtain amino-modified Fe3O4@MoS2. The infrared spectrogram is as Figure 3 shown in C. The characteristic peak around 562 cm -1 is attributed to the stretching vibration of Fe-O, and the peak at 1633 cm -1 is attributed not only to the bending vibration of the O-H group but also to the deformation of N-H. After modifying Fe3O4@MoS2 with APTES, this peak changes little, while the band at 3443 cm -1 is caused by the vibration of the N-H group; the characterization results prove that the amino group has been successfully modified onto the surface of the Fe3O4@MoS2 material.

[0069] (3) Preparation of UCNPs-Apt

[0070] First, synthesize avidin-modified UCNPs: Take 10 mg of amino-modified upconversion nanomaterials and add them to 5 mL of HEPES buffer (10 mM), ultrasonically disperse for 10 min, and then mix and react with 1.25 mL of glutaraldehyde solution (25%). After the reaction, collect the upconversion nanomaterials by centrifugation (9000 rpm, 3 min), and wash the precipitate with HEPES buffer. Redisperse the obtained precipitate in 5 mL of HEPES buffer, add 1 mg / mL avidin to it, and incubate with shaking at 37 °C for 12 h. Centrifuge (9000 rpm, 3 min) to collect the precipitate, wash the precipitate, and resuspend it in 5 mL of HEPES buffer to obtain 2 mg / mL UCNPs-Avidin.

[0071] Next, prepare aptamer (Apt)-modified UCNPs: Take 5 mL of UCNPs-Avidin (2 mg / mL), disperse it evenly, add biotin-modified methyl parathion aptamer solution (Biotin-Apt, 10 μM), incubate with shaking at 37 °C for 6 h, then centrifuge (9000 rpm, 3 min) and wash to remove unbound Biotin-Apt. Finally, disperse the obtained UCNPs-Apt in 10 mL of HEPES buffer to obtain UCNPs-Apt (1 mg / mL). The characterization results are as Figure 4 shown in Figure A. Compared with UCNPs, the UV-vis absorption spectrum of UCNPs-Apt has an obvious absorption peak at 260 nm, which is the characteristic absorption peak of Apt, proving the successful modification of Apt.

[0072] Among them, the sequence of biotin-modified methyl parathion aptamer (SEQ ID NO.1) is: 5'-Biotin-ATTGGCACTCCACGCATAGGGAGGTCACACCGATAGTGGCGCAATCTAGTTGGTTAGCG CCCTATGCGTGCTACCGTGAA-3', and the sequence length is 80 nt.

[0073] (4) Preparation of Fe3O4@MoS2-cDNA

[0074] First, synthesize Avidin-modified Fe3O4@MoS2: Take 10 mg of amino-modified Fe3O4@MoS2 and add it to 5 mL of HEPES buffer (10 mM), disperse it by ultrasonic wave, then add 1.25 mL of 25% glutaraldehyde solution, and the mixture reacts with shaking at room temperature. After completion, collect the precipitate by magnetic separation, wash it with HEPES buffer, redisperse the precipitate in 5 mL of HEPES buffer, add 1 mg / mL Avidin to it, incubate with shaking at 37 °C for 12 h, then collect the precipitate by magnetic separation, and after washing the precipitate, resuspend it in 5 mL of HEPES to obtain 2 mg / mL of Fe3O4@MoS2-Avidin.

[0075] Next, prepare cDNA-modified Fe3O4@MoS2: Take 5 mL of Fe3O4@MoS2-Avidin (2 mg / mL), disperse it evenly, add biotin-modified methyl parathion aptamer complementary strand solution (Biotin-cDNA, 10 μM), incubate with shaking at 37 °C for 6 h, then collect and wash the precipitate by magnetic separation. Finally, disperse the precipitate in 10 mL of HEPES to obtain Fe3O4@MoS2-cDNA (1 mg / mL). The characterization results are as Figure 4As shown in B, compared with Fe3O4@MoS2, the UV-vis absorption spectrum of Fe3O4@MoS2-cDNA shows an obvious absorption peak at 260 nm, which is the characteristic absorption peak of cDNA, proving the successful modification of cDNA.

[0076] Among them, the sequence of the biotinylated methyl parathion aptamer complementary strand (SEQ ID NO.2) is: 5'-Biotin-CACGCATAGGGCGCTAACCAACTAGATTGCGCCACTATCGGTGTGACCTCCCTATGCGTG-3', and the sequence length is 60 nt.

[0077] (5) Preparation of the upconversion fluorescence sensor

[0078] Mixing UCNPs-Apt with Fe3O4@MoS2-cDNA gives the upconversion fluorescence sensor.

[0079] Example 2

[0080] In order to obtain the best detection effect of methyl parathion in Polygonatum sibiricum, on the basis of Example 1, the detection conditions of the upconversion fluorescence sensor were optimized.

[0081] First, the concentration of Fe3O4@MoS2-cDNA was optimized. 500 μL of UCNPs-Apt (concentration 1 mg / mL) was mixed with 300 μL of Fe3O4@MoS2-cDNA with different concentrations (concentrations were 0.2, 0.4, 0.6, 0.8, 1.0 mg / mL) respectively, then buffer solution (200 μL) was added and shaken well, reacted at 37 °C for 30 min, the fluorescence spectrum was collected, and the best use concentration of Fe3O4@MoS2-cDNA was determined according to the fluorescence intensity.

[0082] Then, the incubation time of Aptamer and cDNA was optimized. UCNPs-Apt (1 mg / mL, 500 μL), Fe3O4@MoS2-cDNA (0.8 mg / mL, 300 μL) and buffer solution (200 μL) were mixed evenly and reacted at 37 °C for different times (5, 10, 20, 30, 40 min) respectively, the upconversion fluorescence spectra at different incubation times were collected, and the best reaction time was determined according to the fluorescence intensity.

[0083] Finally, optimize the binding time of the aptamer with parathion-methyl. Mix UCNPs-Apt (1 mg / mL, 500 μL) with Fe3O4@MoS2-cDNA (0.8 mg / mL, 300 μL) and react at 37 °C for 30 min to construct a detection system. Then add parathion-methyl (1 ng / mL, 200 μL) to the above system and incubate with shaking for different times (10, 15, 20, 30, 40, 50 min), collect the upconversion fluorescence spectrum, and determine the optimal binding time according to the fluorescence intensity.

[0084] According to Figure 5 the optimization results shown, obtain the detection procedure of the upconversion fluorescence sensor: Mix UCNPs-Apt (1 mg / mL, 500 μL) with Fe3O4@MoS2-cDNA (0.8 mg / mL, 300 μL), react at 37 °C for 30 min, then add different concentrations of parathion-methyl (200 μL) and continue to react for 30 min, and collect the fluorescence spectrum under the excitation of a 980 nm exciter.

[0085] Example 3

[0086] To evaluate the detection sensitivity of the constructed upconversion fluorescence sensor, test different concentrations (0.01, 0.1, 1, 10, 100 ng / mL) of parathion-methyl according to the detection procedure described in Example 2. The results are as Figure 6 shown in A, it can be seen that as the concentration of parathion-methyl increases, the difference in fluorescence intensity continuously expands. Figure 6 In B, the relationship between the relative fluorescence intensity value (F0 - F) at 540 nm and the logarithm of the parathion-methyl concentration is shown. It can be seen that in the concentration range of 0.01 - 100 ng / mL, there is a good linear relationship between the relative fluorescence intensity value and the logarithm of the parathion-methyl concentration. The linear regression equation is F0 - F = 279299x - 3×10 6 , and the correlation coefficient R 2 is 0.9934, and the detection limit is 0.002 ng / mL (where F0 and F represent the fluorescence intensities at 540 nm in the absence and presence of parathion-methyl, respectively, and x is the logarithm of the parathion-methyl concentration). This result indicates that the upconversion fluorescence sensor of the present invention has high detection sensitivity for parathion-methyl.

[0087] Example 4

[0088] To evaluate the detection specificity of the constructed upconversion fluorescence sensor, the present invention selects 5 kinds of interferents: bifenthrin (BIF), lambda-cyhalothrin (LCT), flucythrinate (FCT), chlorpyrifos-methyl (CPM), parathion (PT) and compare with parathion-methyl (MP). When testing, the concentration of all pesticides is 10 ng / mL. AsFigure 7 As shown in A, when only one pesticide is added to the sensing system, the up-conversion fluorescence intensity of the sensing system with only parathion-methyl present is significantly lower than that of the sensing systems with other interfering substances present. In addition, as Figure 7 shown in B, after adding parathion-methyl to the detection systems containing other interfering substances respectively, its up-conversion fluorescence intensity has no obvious difference compared with that when parathion-methyl exists alone. These results indicate that the up-conversion fluorescence sensor of the present invention has strong detection specificity for parathion-methyl.

[0089] Example 5

[0090] In order to verify the feasibility of the constructed up-conversion fluorescence sensor for detecting parathion-methyl in polygonatum sibiricum, the standard addition method was used for the spike recovery experiment. The specific operation process is as follows:

[0091] (1) Pretreat the polygonatum sibiricum sample: First, wash the rhizome of polygonatum sibiricum clean, place it in a constant temperature blast drying oven, continuously dehydrate and dry it at 60 °C, then transfer it to a crusher for intermittent grinding (60 s each time, cooling for 2 min at intervals, circulating 2 times), and sieve it through a 100-mesh sieve to obtain polygonatum sibiricum powder. Add 100 μL of parathion-methyl standard products with known concentrations (0.1, 1, and 10 μg / mL) to 2 g of polygonatum sibiricum powder respectively. Mix the prepared spiked samples with 5 mL of acetonitrile, vortex for 5 min, centrifuge (6000 rpm, 10 min) to remove the precipitate, repeat the above steps, and combine the supernatant. Then place the supernatant in a rotary evaporator at 80 °C to heat until the acetonitrile solvent evaporates nearly dry. Then dissolve the dried residue in 10 mL of methanol solution and vortex to obtain the parathion-methyl solution to be detected for quantitative detection.

[0092] (2) Mix UCNPs-Apt (1 mg / mL, 500 μL) with Fe3O4@MoS2-cDNA (0.8 mg / mL, 300 μL), react at 37 °C for 30 min, then add the parathion-methyl solution to be detected (200 μL) and continue to react for 30 min. Collect the fluorescence spectrum under the excitation of a 980 nm exciter to obtain the up-conversion fluorescence intensity value at 540 nm, substitute it into the standard curve described in Example 3, and calculate the content of parathion-methyl in polygonatum sibiricum.

[0093] To further verify the accuracy and reliability of the constructed upconversion fluorescence sensor for the detection of parathion-methyl in polygonatum sibiricum, the enzyme-linked immunosorbent assay (ELISA) was simultaneously used to determine the content of parathion-methyl in polygonatum sibiricum samples. By comparing the detection results of the two methods, Table 1 shows that the detection results of the two are relatively close. In addition, a two-tailed paired T-test was performed, and the calculated P value was greater than the significance factor of 0.05, indicating that there was no significant difference in the detection results of the two methods. The above results prove that the upconversion fluorescence sensor constructed in the present invention is accurate and reliable in detection and has application value for the detection of parathion-methyl in polygonatum sibiricum.

[0094] Table 1 Detection results of parathion-methyl in polygonatum sibiricum samples

[0095]

[0096] a SD: Standard deviation, n = 3; b RSD: Relative standard deviation; c P: P>0.05 indicates no significant difference;

[0097] The detected amount of parathion-methyl in polygonatum sibiricum: X = (C × V × 10 -3 ) / m

[0098] Where X: the detected amount of parathion-methyl in polygonatum sibiricum, in milligrams per kilogram (mg / kg); C: the detected concentration of parathion-methyl in polygonatum sibiricum, in nanograms per milliliter (ng / mL); V: the volume of constant volume, in milliliters (mL); m: the mass of polygonatum sibiricum weighed, in grams (g).

[0099] The above-described embodiments are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An upconversion fluorescence sensor for detecting methyl parathion in polygonatum sibiricum, characterized in that, The upconversion fluorescence sensor includes an upconversion nanomaterial modified with methyl parathion aptamer and an Fe3O4@MoS2 nanocomposite modified with cDNA, wherein the nucleotide sequences of the methyl parathion aptamer and the cDNA are complementary and paired, and the upconversion nanomaterial is a rare earth upconversion nanomaterial doped with gadolinium, ytterbium and / or erbium.

2. The upconversion fluorescence sensor according to claim 1, wherein, The nucleotide sequence of the methyl parathion aptamer is shown in SEQ ID NO: 1, and the nucleotide sequence of the cDNA is shown in SEQ ID NO:

2.

3. A preparation method of an upconversion fluorescence sensor for detecting methyl parathion in polygonatum sibiricum, which is characterized in that, It includes the following steps: (1) Dissolve polyethyleneimine in ethylene glycol, then add gadolinium chloride hexahydrate, sodium chloride, erbium chloride anhydrous and ytterbium chloride hexahydrate to react to obtain a mixed solution. Dissolve ammonium chloride in ethylene glycol and then drop it into the mixed solution to continue the reaction. Then transfer it to a reaction kettle for high-temperature reaction to obtain an amino-modified upconversion nanomaterial; (2) Mix ferric chloride, anhydrous sodium acetate and ethylene glycol to react to obtain Fe3O4 nanoparticles. Then ultrasonically disperse the Fe3O4 nanoparticles in distilled water, add ammonium molybdate tetrahydrate and thiourea for high-temperature reaction to obtain an Fe3O4@MoS2 nanocomposite; Mix the Fe3O4@MoS2 nanocomposite with 3-aminopropyltriethoxysilane in an ethanol solution and oscillate to react to obtain an amino-modified Fe3O4@MoS2; (3) Mix and oscillate the amino-modified upconversion nanomaterial with avidin for incubation to obtain an avidin-modified upconversion nanomaterial; Then mix and oscillate the avidin-modified upconversion nanomaterial with a biotin-modified methyl parathion aptamer for incubation to obtain a methyl parathion aptamer-modified upconversion nanomaterial; (4) Mix and oscillate the amino-modified Fe3O4@MoS2 with avidin for incubation to obtain an avidin-modified Fe3O4@MoS2; Then mix and oscillate the avidin-modified Fe3O4@MoS2 with a biotin-modified cDNA for incubation to obtain a cDNA-modified Fe3O4@MoS2 nanocomposite; (5) Mix and incubate the methyl parathion aptamer-modified upconversion nanomaterial and the cDNA-modified Fe3O4@MoS2 nanocomposite to obtain an upconversion fluorescence sensor for detecting methyl parathion in polygonatum sibiricum.

4. The preparation method according to claim 3, characterized in that, In step (1), the dosage ratio of the polyethyleneimine, ethylene glycol, gadolinium chloride hexahydrate, sodium chloride, erbium chloride anhydrous and ytterbium chloride hexahydrate is 500 mg: 35 mL: 361.1 mg: 58.5 mg: 13.3 mg: 76.4 mg, and the reaction conditions are: stir for 20 - 30 min; The dosage ratio of the ammonium fluoride and ethylene glycol is 296.3 mg: 15 mL, and the reaction conditions are: stir for 8 - 12 min; The conditions for the high-temperature reaction in the reaction kettle are: react at 180 - 200 °C for 10 - 12 h.

5. The preparation method according to claim 3, characterized in that, In step (2), the dosage ratio of the ferric chloride, anhydrous sodium acetate and ethylene glycol is 0.5 g: 3.0 g: 30 mL, and the reaction conditions are: react at 180 - 190 °C for 4 h; The dosage ratio of the Fe3O4 nanoparticles, ammonium molybdate tetrahydrate and thiourea is 30 mg: 0.8 g: 2.0 g, and the high-temperature reaction conditions are: reacting at 180 - 190 °C for 12 h; The dosage ratio of the Fe3O4@MoS2 nanocomposite and 3-aminopropyltriethoxysilane is 40 mg: 500 μL, and the conditions for the oscillating reaction are: oscillating reaction at 60 °C for 12 h.

6. The preparation method according to claim 3, characterized in that, In step (3), the temperature for mixing and oscillating incubation of the amino-modified upconversion nanomaterial and avidin is 37 °C, and the time is 12 h; The temperature for mixing and oscillating incubation of the avidin-modified upconversion material and the biotin-modified methyl parathion aptamer is 37 °C, and the time is 6 h; The nucleotide sequence of the biotin-modified methyl parathion aptamer is as shown in SEQ ID NO:

1.

7. The preparation method according to claim 3, characterized in that In step (4), the temperature for mixing and oscillating incubation of the amino-modified Fe3O4@MoS2 and avidin is 37 °C, and the time is 12 h; The temperature for mixing and oscillating incubation of the avidin-modified Fe3O4@MoS2 and the biotin-modified cDNA is 37 °C, and the time is 6 h; The nucleotide sequence of the biotin-modified cDNA is as shown in SEQ ID NO:

2.

8. The preparation method according to claim 3, characterized in that, In step (5), the volume ratio of the methyl parathion aptamer-modified upconversion nanomaterial and the cDNA-modified Fe3O4@MoS2 nanocomposite is 5:3, the concentration of the methyl parathion aptamer-modified upconversion nanomaterial is 1 mg / mL, the concentration of the cDNA-modified Fe3O4@MoS2 nanocomposite is 0.8 mg / mL, and the conditions for the mixed incubation are: reacting at 37 °C for 30 min.

9. Use of the upconversion fluorescence sensor according to claim 1 or 2 in detecting methyl parathion in Polygonatum sibiricum.

10. A method for detecting parathion-methyl in polygonatum sibiricum, characterized in that, It includes the step of mixing and reacting the upconversion fluorescence sensor according to claim 1 or 2 with the Polygonatum sibiricum sample to be tested, and obtaining the methyl parathion content in Polygonatum sibiricum by detecting the upconversion fluorescence intensity value at 540 nm.

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